Anti-tetrodotoxin antibody y126c, and preparation method and use thereof

ABSTRACT

The present disclosure relates to the technical field of biomedicine, and provides an anti-tetrodotoxin humanized antibody Y126C and use thereof. The humanized antibody has a heavy-chain variable region and a light-chain variable region with amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 2, respectively. Affinity analysis shows that the antibody of the present disclosure has prominent affinity. It is proved by experiments that, after mice in an antibody protection group pre-injected with the antibody of the present disclosure are injected with tetrodotoxin, most of mice do not show toxic symptoms, and during continuous observation for one month, no toxic lethality occurs, indicating that the antibody of the present disclosure shows excellent anti-tetrodotoxin effects, excellent preventive or therapeutic effects on puffer fish-related biological injuries, and promising clinical application prospects.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to the Chinese Patent Application No.202111601192.8 with a filing date of Dec. 24, 2021. The content of theaforementioned application, including any intervening amendmentsthereto, is incorporated herein by reference.

CROSS-REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 0601SEQ list.xml, created on May. 29, 2023, with asize of 3,094 bytes. The Sequence Listing is incorporated by referenceherein.

TECHNICAL FIELD

The present disclosure belongs to the technical field of biomedicine,and specifically relates to an anti-tetrodotoxin humanized antibodyY126C, a preparation method and use thereof in the preparation oftetrodotoxin formulations.

BACKGROUND

The puffer meat is tender and delicious. The fish skin with thorns has athick and gelatinous texture, which is sticky and tastes far better thanshark fins and sea cucumbers. Therefore, puffer fish is known as thefirst of the “Three Fresh Dishes on the Yangtze River”. However, mostpuffer fish contain tetrodotoxin (TTX) concentrated in the ovary, liver,kidney, blood, eyes, gills, and skin. Tetrodotoxin is relatively stable,and is not easy to be eliminated by salting, sun exposure, and generalheating and cooking. The chemical research of tetrodotoxin began in1909. After 1964, Woodward determined the structure of TTX. In 1972,Kishi et al. successfully synthesized tetrodotoxin by chemical methods.Tetrodotoxin has a molecular formula of C₁₁H₁₇N₃O₈ and a molecularweight of 319.27; the tetrodotoxin molecule is mainly composed of threenitrogen atoms, which form a special structure with the hydrogen andoxygen atoms. The tetrodotoxin molecule includes one carbocyclic ring,one guanidino group, six hydroxyl groups, and a separate ring ligated bysemi-aldose lactone at the C-5 and C-10 positions. Tetrodotoxin is alsoone of the deadliest poisons in the world, and is called by some experts“one of nature's strangest molecules”. 1 g of tetrodotoxin is 10,000times more toxic than 1 g of cyanide.

Because antibodies can efficiently and specifically bind to variousantigen proteins in vivo and in vitro, antibodies can not only be usedto regulate the functions of immune systems, but also can be used forvarious detection methods with high sensitivity. At present, antibodydrugs are the most important part of biotechnology drugs, and antibodyreagents are also one of the most common reagents used in medicaldiagnosis and biological research. Therefore, antibody-relatedbiological products have extremely-promising application prospects andextremely-high commercial values. Antibodies can be obtained in avariety of ways. For toxin proteins, antibodies are highly neutralizingas a potential antitoxin drug.

SUMMARY

The present disclosure is intended to study an anti-tetrodotoxinantibody Y126C, and a preparation method and use thereof based on theabove research background, that is, to provide a brand-new humanizedantibody, and a preparation method and use thereof.

In a first aspect of the present disclosure, an anti-tetrodotoxinhumanized antibody Y126C is provided, where the heavy-chain variableregion of the antibody Y126C is composed of anFRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 region; and the light-chainvariable region of the antibody Y126C is composed of anFRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 region.

The heavy-chain variable region and the light-chain variable region haveamino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2,respectively.

The heavy-chain variable region has the amino acid sequence (SEQ IDNO: 1) as follows:

EVQLVESGGGLVQPGRSLRLSCAASGMMAHWAAAMHWVRQAPGK GLEWVSAMAKQYRNRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVLLK HQMLDYWGQGTLVTVSS

The light-chain variable region has the amino acid sequence (SEQ ID NO:2) as follows:

DIQMTQSPSSLSASVGDRVTITCRAVLFMVIYYATWYQQKPGQAPILV IYGYQQVMQGIPDRF SGS SSGNTASLTITGAQAEDEADYYCIVKMFEMFGQG TKVEIK

The humanized antibody can be obtained as follows: a humanized antibodyphage display library is first constructed, then the humanized antibodyis screened, specific positive clones are screened by phageenzyme-linked immunosorbent assay (ELISA), and after sequence analysis,the humanized antibody is obtained. After expressing and purifying thehumanized antibody in a 293 system, high-purity humanized antibodies areobtained.

In a second aspect of the present disclosure, a preparation method ofthe humanized antibody is provided, including the following steps:

-   -   (A) synthesizing a full-length humanized antibody based on a        whole genome of a variable region gene of the antibody Y126C;    -   (B) cloning the full-length humanized antibody obtained in        step (A) into an expression vector by PCR, and determining a        correct clone after sequencing verification; and    -   (C) introducing the expression vector into a host cell for        fusion protein expression.

In the present disclosure, any suitable vectors are applicable, whichmay preferably be pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDHFR, orpDR1; and the expression vector may include a fusion DNA sequenceligated with appropriate transcription and translation regulatorysequences.

In the present disclosure, a mammalian or insect host cell or aprokaryotic cell culture system can be used for the expression of thefusion protein of the present disclosure. An available host cell may bea prokaryotic cell with the above-mentioned vector, which can be onefrom the group consisting of DH5a, Top10, BL21 (DE3), and TG1.

The fusion protein of the present disclosure can be easily produced inthe following cells: mammalian cells, such as CHO, NSO, HEK293, BHK, orCOS cells; bacterial cells, such as E. coli, Bacillus subtilis (B.subtilis), or Pseudomonas fluorescens (P. fluorescens); and insectcells, or fungal or yeast cells, which are cultivated using techniquesknown in the art.

The preparation method of the fusion protein disclosed in the presentdisclosure may include: cultivating the above-mentioned host cell underexpression conditions to express, isolate, and purify the fusionprotein. With the above method, the antibody can be purified into asubstantially homogeneous substance, such as a single band of sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

The fusion protein disclosed in the present disclosure can be isolatedand purified by affinity chromatography. According to characteristics ofan affinity column used, a conventional method such as high-salt bufferand pH change can be used to elute the fusion protein peptide bound tothe affinity column.

Various protein purification methods can be used, and such methods areknown in the art and described in, for example, (Wilchek and Bayer,1990, Methods in enzymology) (Scopes, 2013, Protein purification:principles and practice).

According to Biacore analysis, the humanized antibody of the presentdisclosure has prominent affinity, and small animal experiments haveshown that, after mice in a protection group pre-injected with thehumanized antibody of the present disclosure are injected withtetrodotoxin, no mice shows neurotoxic symptoms, and during continuousobservation for one month, no toxic lethality occurs. It indicates thatthe humanized antibody of the present disclosure has an excellentanti-tetrodotoxin effect.

Therefore, in a third aspect of the present disclosure, a pharmaceuticalcomposition with the humanized antibody is provided. In addition to thehumanized antibody, the pharmaceutical composition may include apharmaceutically acceptable drug carrier.

The humanized antibody of the present disclosure and a pharmaceuticallyacceptable adjuvant together constitute a pharmaceutical formulationcomposition, thereby exerting a more stable therapeutic effect. Theformulation can ensure the conformational integrity of an amino acidcore sequence of the humanized antibody disclosed in the presentdisclosure, and can also protect multifunctional groups of the proteinto prevent its degradation (including but not limited to aggregation,deamination, or oxidation).

Normally, a liquid preparation can be stored at 2° C. to 8° C. for atleast one year, and a lyophilized formulation can be stored at 30° C.for at least six months. The formulation can be a suspension, aninjection, a lyophilized formulation, or the like commonly used in thepharmaceutical field, and preferably an injection or a lyophilizedformulation.

For the injection or lyophilized formulation of the humanized antibodydisclosed in the present disclosure, the pharmaceutically acceptableadjuvant may include one or a combination of two or more from the groupconsisting of a surfactant, a solution stabilizer, an isoosmoticadjusting agent, and a buffer. The surfactant may include a non-ionicsurfactant, such as polyoxyethylene sorbitan fatty acid esters (Tween 20or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecylsulfate (SDS), sodium lauryl sulfate (SLS); myristyl, linoleyl orstearyl sarcosine; Pluronics; and MONAQUATTM; and the surfactant may beadded at an amount that minimizes the granulation tendency of thebifunctional bispecific antibody protein. The solution stabilizer can besugar, including reducing sugar and non-reducing sugar; amino acid,including monosodium glutamate (MSG) or histidine; and alcohols,including one or a combination of two or more from the group consistingof triol, higher sugar alcohol, propylene glycol (PG), and polyethyleneglycol (PEG); and the solution stabilizer may be added at an amount thatenables a final formulation to remain stable within a period of timeconsidered by those skilled in the art to reach a stable state. Theisoosmotic adjusting agent can be one from the group consisting ofsodium chloride and mannitol. The buffer can be one from the groupconsisting of tris(hydroxymethyl)aminomethane (TRIS), a histidinebuffer, and a phosphate buffer.

The above-mentioned formulation is a composition including the humanizedantibody, and after being administered to animals including humans, theformulation shows a prominent anti-tetrodotoxin effect. Specifically,the formulation is effective in preventing and/or treating tetrodotoxinpoisoning, and can be used as an anti-tetrodotoxin drug.

In the present disclosure, when the humanized antibody and thecomposition thereof are administered to animals including humans, adosage varies with the age and body weight of the patient, thecharacteristics and severity of the disease, and the route ofadministration. The total dosage can be defined within a specified rangewith reference to results of animal experiments and various otherconditions. Specifically, a dosage of intravenous injection may be 1mg/d to 1,800 mg/d.

In a fourth aspect of the present disclosure, use of the humanizedantibody (specifically use in the preparation of an anti-tetrodotoxinformulation drug) for preventing or treating jellyfish stings isprovided.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following examples and experimental examples are provided to furtherillustrate the present disclosure, and shall be construed as alimitation to the present disclosure. The examples do not includedetailed descriptions of traditional methods, such as methods forconstructing vectors and plasmids, methods for inserting genes encodingproteins into such vectors and plasmids, or methods for introducingplasmids into host cells. Such methods are well known to those ofordinary skill in the art, and are described in many publications,including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) MolecularCloning: A Laboratory Manual, 2^(nd) edition, Cold spring HarborLaboratory Press.

Example 1. Preparation and Expression of a Humanized Antibody

The humanized antibody was derived from a phage-displayed antibodylibrary. The methods for constructing and expressing the antibody wereconventional experimental techniques in the field, which were brieflydescribed as follows:

-   -   (1) A heavy chain and a light chain of the antibody were        synthesized by whole genome, where amino acid sequences of a        heavy-chain variable region and a light-chain variable region        were shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.    -   (2) Expression and purification of the antibody

The expression and purification of the antibody were conducted accordingto a method in the literature [Hu S, Fu W, Li T, et al. Antagonism ofEGFR and Notch limits resistance to EGFR inhibitors and radiation bydecreasing tumor-initiating cell frequency [J]. Science TranslationalMedicine, 2017, 9 (380)], and a purity of the antibody was identified bySDS-PAGE and reached more than 95%.

Example 2. Biacore Analysis

An anti-tetrodotoxin antibody was coated on a CM5M5 chip (GE), and afterthe antibody to be tested was captured, the affinity of each fusionprotein was detected by Biacore T100 (GE Healthcare). Specific detectedaffinity values were shown in Table 1.

TABLE 1 Biacore analysis results Parameter Unit Bindingaffinity/kinetics KD (nM) 70.6

Example 3. Small Animal Experiment

32 C57 mice with a body weight of (20±2) g were selected and fasted for12 h before the experiment (without water deprivation). The mice wererandomly divided into three groups (half female and half male for eachgroup): half-lethal dosage tetrodotoxin group: 12 mice; drug protectiongroup: 10 mice, which were pre-injected with the humanized antibody at10 mg/kg; and blank control group: 10 mice, which were administered withnormal saline. The mice were administered intraperitoneally. Within 1 hafter the administration, the mice in the blank control group all showedtypical neurotoxic symptoms. Only one of the mice in the antibodyprotection group showed neurotoxic symptoms, and during continuousobservation for one month, no toxic lethality occurred. Specific resultswere shown in Table 2.

TABLE 2 Test results of the anti-tetrodotoxin effect of the antibodyNumber of Number of survivors on survivors Total the day after one monthHumanized antibody 10 9 9 Control group 10 0 0

The preferred examples of the present disclosure have been described indetail above, but the present disclosure is not limited to theseexamples. Those skilled in the art can make various equivalentvariations or substitutions without departing from the spirit of thepresent disclosure, and these equivalent variations or substitutions areall included in the scope defined by the claims of this application.

What is claimed is:
 1. An anti-tetrodotoxin humanized antibody Y126C,comprising a heavy-chain variable region with an amino acid sequenceshown in SEQ ID NO: 1, and a light-chain variable region with an aminoacid sequence shown in SEQ ID NO:
 2. 2. The anti-tetrodotoxin antibodyY126C according to claim 1, wherein the heavy-chain variable regioncomprises FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 regions; and thelight-chain variable region comprisesFRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 regions.
 3. A nucleotide encodingthe anti-tetrodotoxin antibody Y126C according to claim
 1. 4. Apreparation method of the anti-tetrodotoxin antibody Y126C according toclaim 1, comprising the following steps: (A) synthesizing a full-lengthhumanized antibody based on a whole genome of a variable region gene ofthe antibody Y126C; (B) cloning the full-length humanized antibodyobtained in step (A) into an expression vector by PCR, and determining acorrect clone after sequencing verification; and (C) introducing theexpression vector into a host cell for fusion protein expression.
 5. Thepreparation method according to claim 4, wherein the expression vectoris pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDHFR, or pDR1; theexpression vector comprises a fusion DNA sequence ligated withappropriate transcription and translation regulatory sequences; and thehost cell is a prokaryotic cell, a mammalian cell, a bacterial cell, aninsect cell, or a fungal cell.
 6. A pharmaceutical compositioncomprising the anti-tetrodotoxin antibody Y126C according to claim 1,wherein the pharmaceutical composition further comprises apharmaceutically acceptable drug carrier.
 7. The pharmaceuticalcomposition according to claim 6, wherein the pharmaceutical compositionis an injection or a lyophilized formulation; and the pharmaceuticallyacceptable drug carrier comprises one or a combination of two or morefrom the group consisting of a surfactant, a solution stabilizer, anisoosmotic adjusting agent, and a buffer.
 8. The pharmaceuticalcomposition according to claim 6, wherein the injection or thelyophilized formulation is intravenously injected at a dosage of 1 mg/dto 1,800 mg/d.
 9. Use of the anti-tetrodotoxin antibody Y126C accordingto claim 1 in preparation of an anti-tetrodotoxin drug.